U.S. patent number 6,552,065 [Application Number 09/944,275] was granted by the patent office on 2003-04-22 for deacetylase inhibitors.
This patent grant is currently assigned to Novartis AG. Invention is credited to Kenneth Walter Bair, Michael Alan Green, Lawrence Blas Perez, Stacy William Remiszewski, Lidia Cristina Sambucetti, Sushil Sharma, Richard William Versace.
United States Patent |
6,552,065 |
Remiszewski , et
al. |
April 22, 2003 |
**Please see images for:
( Certificate of Correction ) ** |
Deacetylase inhibitors
Abstract
The present invention provides hydroxamate compounds which are
deacetylase inhibitors. The compounds are suitable for
pharmaceutical compositions having anti-proliferative
properties.
Inventors: |
Remiszewski; Stacy William
(Washington Township, NJ), Bair; Kenneth Walter (Mountain
Lakes, NJ), Versace; Richard William (Wanaque, NJ),
Perez; Lawrence Blas (Hackettstown, NJ), Green; Michael
Alan (Easton, PA), Sambucetti; Lidia Cristina (Pacifica,
CA), Sharma; Sushil (West Orange, NJ) |
Assignee: |
Novartis AG (Basel,
CH)
|
Family
ID: |
26923760 |
Appl.
No.: |
09/944,275 |
Filed: |
August 31, 2001 |
Current U.S.
Class: |
514/416; 514/339;
546/277.1; 548/470; 548/506; 514/419 |
Current CPC
Class: |
A61P
43/00 (20180101); C07D 405/12 (20130101); C07D
209/16 (20130101); C04B 35/632 (20130101); C07D
401/12 (20130101); A61P 35/00 (20180101); C07D
307/81 (20130101); C07D 403/12 (20130101); C07D
471/04 (20130101); C07D 233/64 (20130101); C07C
259/06 (20130101); C07D 295/155 (20130101) |
Current International
Class: |
C07D
401/00 (20060101); C07D 295/155 (20060101); C07D
233/54 (20060101); C07D 401/12 (20060101); C07D
209/16 (20060101); C07D 295/00 (20060101); C07D
403/00 (20060101); C07C 259/00 (20060101); C07C
259/06 (20060101); C07D 471/04 (20060101); C07D
471/00 (20060101); C07D 405/00 (20060101); C07D
403/12 (20060101); C07D 209/00 (20060101); C07D
233/00 (20060101); C07D 307/81 (20060101); C07D
405/12 (20060101); C07D 307/00 (20060101); A61K
031/40 (); A61K 031/403 (); C07D 209/44 (); C07D
401/04 () |
Field of
Search: |
;514/419,339,416
;548/506,470 ;546/277.1 |
Foreign Patent Documents
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Jul 1998 |
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JP |
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WO 93/12075 |
|
Jun 1993 |
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WO |
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WO 95/31977 |
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Nov 1995 |
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WO |
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WO 97/35990 |
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Oct 1997 |
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WO |
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WO 98/55449 |
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Dec 1998 |
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WO |
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WO 00/23112 |
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Apr 2000 |
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WO |
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WO 01/18171 |
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Mar 2001 |
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WO |
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WO 01/38322 |
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May 2001 |
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WO |
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WO 01/42437 |
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Jun 2001 |
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WO |
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WO 01/70675 |
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Sep 2001 |
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WO |
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Other References
M Yoshida et al., "Histone deacetylase as a new target for cancer
chemotherapy" Cancer Chemother. Pharmacol., vol. 48, Suppl. 1, Jul.
1, 2001, pp. S20-S-26, XP-002188621. .
Buggy et al., "Cloning and characterization of a novel human
histone deacetylase, HDAC8," Biochemical Society, vol. 350, pp.
199-205 (2000). .
Kim et al., "Oxamflatin is a novel antitumor compound that inhibits
mammalian histone deacetylase," Oncogene, vol. 18, pp. 2461-2470
(1999). .
Lavoie et al., "Design and Synthesis of a Novel Class of Histone
Deacetylase Inhibitors," Bioorg. & Med.Chem.Letters, vol. 11,
pp. 2847-2850 (2001). .
Taunton et al., "Synthesis of Natural and Modified Trapoxins,
Useful Reagents for Exploring Histone Deacetylase Function,"
American Chemical Society, vol. 118, pp. 10412-10422
(1996)..
|
Primary Examiner: McKane; Joseph K.
Assistant Examiner: Wright; Sonya
Attorney, Agent or Firm: Dohmann; George R.
Parent Case Text
This application claims the benefit of Provisional Application No.
60/307,490 filed Jul. 24, 2001, Provisional Application No.
60/292,232 filed May 18, 2001, and Provisional Application No.
60/229,943 filed Sep. 1, 2000, the disclosure of which is herein
incorporated by reference.
Claims
What is claimed is:
1. A compound of the formula (I) ##STR309##
wherein R.sub.1 is H, halo, or a straight chain C.sub.1 -C.sub.6
alkyl; R.sub.2 is selected from H, C.sub.1 -C.sub.10 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, C.sub.4
-C.sub.9 heterocycloalkylalkyl, cycloalkylalkyl, aryl, heteroaryl,
arylalkyl, heteroarylalkyl, --(CH.sub.2).sub.n C(O)R.sub.6,
--(CH.sub.2).sub.n OC(O)R.sub.6, amino acyl,
HON--C(O)--CH.dbd.C(R.sub.1)-aryl-alkyl- and --(CH.sub.2).sub.n
R.sub.7 ; R.sub.3 and R.sub.4 are the same or different and
independently H, C.sub.1 -C.sub.6 alkyl, acyl or acylamino, or
R.sub.3 and R.sub.4 together with the carbon to which they are
bound represent C.dbd.O, C.dbd.S, or C.dbd.NR.sub.8 ; R.sub.5 is
selected from polyheteroaryl which is substituted or unsubstituted
indol-3-yl; n, n.sub.1, n.sub.2 and n.sub.3 are the same or
different and independently selected from 0-6, when n, is 1-6, each
carbon atom can be optionally and independently substituted with
R.sub.3 and/or R.sub.4 ; X and Y are the same or different and
independently selected from H, halo, C.sub.1 -C.sub.4 alkyl,
NO.sub.2, C(O)R.sub.1, OR.sub.9, SR.sub.9, CN, and NR.sub.10
R.sub.11 ; R.sub.6 is selected from H, C.sub.1 -C.sub.6 alkyl,
C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl,
cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,
OR.sub.12, and NR.sub.13 R.sub.14 ; R.sub.7 is selected from
OR.sub.15, SR.sub.15, S(O)R.sub.16, SO.sub.2 R.sub.17, NR.sub.13
R.sub.14, and NR.sub.12 SO.sub.2 R.sub.6 ; R.sub.8 is selected from
H, OR.sub.15, NR.sub.13 R.sub.14, C.sub.1 -C.sub.6 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl,
heteroaryl, arylalkyl, and heteroarylalkyl; R.sub.9 is selected
from C.sub.1 -C.sub.4 alkyl and C(O)-alkyl; R.sub.10 and R.sub.11
are the same or different and independently selected from H,
C.sub.1 -C.sub.4 alkyl, and --C(O)-alkyl; R.sub.12 is selected from
H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4
-C.sub.9 heterocycloalkyl, C.sub.4 -C.sub.9 heterocycloalkylalkyl,
aryl, mixed aryl and non-aryl polycycle, heteroaryl, arylalkyl, and
heteroarylalkyl; R.sub.13 and R.sub.14 are the same or different
and independently selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, amino acyl, or R.sub.13 and
R.sub.14 together with the nitrogen to which they are bound are
C.sub.4 -C.sub.9 heterocycloalkyl, heteroaryl, polyheteroaryl,
non-aromatic polyheterocycle or mixed aryl and non-aryl
polyheterocycle; R.sub.15 is selected from H, C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and
(CH.sub.2).sub.m ZR.sub.12 ; R.sub.16 is selected from C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, polyheteroaryl, arylalkyl,
heteroarylalkyl and (CH.sub.2).sub.m ZR.sub.12 ; R.sub.17 is
selected from C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl,
C.sub.4 -C.sub.9 heterocycloalkyl, aryl, aromatic polycycle,
heteroaryl, arylalkyl, heteroarylalkyl, polyheteroaryl and
NR.sub.13 R.sub.14 ; m is an integer selected from 0 to 6; and Z is
selected from O, NR.sub.13, S and S(O);
or a pharmaceutically acceptable salt thereof.
2. A compound of claim 1 wherein each of R.sub.1, X, Y, R.sub.3,
and R.sub.4 is H.
3. A compound of claim 2 one of n.sub.2 and n.sub.3 is zero and the
other is 1.
4. A compound of claim 3 wherein R.sub.2 is H or --CH.sub.2
--CH.sub.2 --OH.
5. A compound of claim 1 of the formula (Ia) ##STR310##
wherein n.sub.4 is 0-3, R.sub.2 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, --(CH.sub.2).sub.n C(O)R.sub.6, amino acyl and
--(CH.sub.2).sub.n R.sub.7 ; R.sub.5 ' is polyheteroaryl which is
substituted or unsubstituted indol-3-yl,
or a pharmaceutically acceptable salt thereof.
6. A compound of claim 1 of formula Ib: ##STR311##
wherein R.sub.2 ' is selected from H, C.sub.1 -C.sub.6 alkyl,
C.sub.4 -C.sub.6 cycloalkyl, alkylcycloalkyl, and
(CH.sub.2).sub.2-4 OR.sub.21 where R.sub.21 is H, methyl, ethyl,
propyl, or isopropyl, and R.sub.5 " is unsubstituted or substituted
1H-indol-3-yl,
or a pharmaceutically acceptable salt thereof.
7. A compound of claim 1 of formula (Id) ##STR312##
wherein Z.sub.1 is N--R.sub.20 ; R18 is H, halo, C.sub.1 -C.sub.6
alkyl, C.sub.3 -C.sub.7 cycloalkyl, aryl, or heteroaryl; R.sub.20
is H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkyl-C.sub.3
-C.sub.9 cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,
acyl or sulfonyl; A, is 1, 2 or 3 substituents which are
independently H, C.sub.1 --C.sub.6 alkyl, --OR.sub.19, halo,
alkylamino, aminoalkyl, halo, or heteroarylalkyl, R.sub.2 is
selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9
cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, alkylcycloalkyl,
aryl, heteroaryl, arylalkyl, heteroarylalkyl, --(CH.sub.2).sub.n
C(O)R.sub.6, amino acyl and --(CH.sub.2).sub.n R.sub.7 ; R.sub.19
is selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9
cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl, heteroaryl,
arylalkyl, and heteroarylalkyl; p is 0-3, and q is 1-5 and r is 0
or q is 0 and r is 1-5
or a pharmaceutically acceptable salt thereof.
8. A compound of claim 7 wherein R.sub.2 is H or --CH.sub.2
--CH.sub.2 --OH and the sum of q and r is 1.
9. A compound of claim 1 of the formula (Id) ##STR313##
wherein Z.sub.1 is N--R.sub.20, R18 is H, halo, C.sub.1 -C.sub.6
alkyl, C.sub.3 -C.sub.7 cycloalkyl, unsubstituted phenyl,
substituted phenyl, or heteroaryl, R.sub.20 is H, C.sub.1 -C.sub.6
alkyl, C.sub.1 -C.sub.6 alkyl-C.sub.3 -C.sub.9 cycloalkyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, acyl or sulfonyl; A.sub.1
is 1, 2 or 3 substituents which are independently H, C.sub.1
-C-.sub.6 alkyl, --OR.sub.19, or halo, R.sub.19 is selected from H,
C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4
-C.sub.9 heterocycloalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl and --(CH.sub.2
CH.dbd.CH(CH.sub.3)(CH.sub.2)).sub.1-3 H; p is 0-3, and q is 1-5
and r is 0 or q is 0 and r is 1-5,
or a pharmaceutically acceptable salt thereof.
10. A compound of claim 9 wherein R.sub.2 is H or --CH.sub.2
--CH.sub.2 --OH and the sum of q and r is 1.
11. A compound of claim 9 wherein R18 is H, fluoro, chloro, bromo,
a C.sub.1 -C.sub.4 alkyl group, a C.sub.3 -C.sub.7 cycloalkyl
group, phenyl or a heteroaryl ring.
12. A compound of claim 9 wherein R.sub.2 is H, or
--(CH.sub.2).sub.s CH.sub.2 OH and wherein s is 1-3.
13. A compound of claim 12 wherein R.sub.1 is H and X and Y are
each H, and wherein q is 1-3 and r is 0 or wherein q is 0 and r is
1-3.
14. A compound of claim 9 wherein R18 is H, methyl, ethyl, t-butyl,
trifluoromethyl, cyclohexyl, phenyl, 4-methoxyphenyl,
4-trifluoromethylphenyl, 2-furanyl, 2-thiophenyl, or 2-, 3- or
4-pyridyl.
15. A compound of claim 14 wherein R.sub.2 is H, or
--(CH.sub.2).sub.5 CH.sub.2 OH and wherein s is 1-3.
16. A compound of claim 15 wherein p is 1-3.
17. A compound of claim 16 wherein R.sub.1 is H and X and Y are
each H, and wherein q is 1-3 and r is 0 or wherien q is 0 and r is
1-3.
18. A compound of claim 17 wherein R.sub.2 H or --CH.sub.2
--CH.sub.2 --OH and the sum of q and r is 1.
19. A compound of claim 9 wherein R.sub.20 is H or C.sub.1 -C.sub.6
alkyl.
20. A compound of claim 9 selected from the group consisting of
N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phe
nyl]-2E-2-propenamide, or a pharmaceutically acceptable salt
thereof.
21. A pharmaceutical composition comprising a pharmaceutically
effective amount of a compound of formula (I) ##STR314##
wherein R.sub.1 is H, halo, or a straight chain C.sub.1 -C.sub.6
alkyl; R.sub.2 is selected from H, C.sub.1 -C.sub.10 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, C.sub.4
-C.sub.9 heterocycloalkylalkyl, cycloalkylalkyl, aryl, heteroaryl,
arylalkyl, heteroarylalkyl, --(CH.sub.2).sub.n C(O)R.sub.6,
--(CH.sub.2).sub.n OC(O)R.sub.6, amino acyl,
HON--C(O)--CH.dbd.C(R.sub.1)-aryl-alkyl- and --(CH.sub.2).sub.n
R.sub.7 ; R.sub.3 and R.sub.4 are the same or different and
independently H, C.sub.1 -C.sub.6 alkyl, acyl or acylamino, or
R.sub.3 and R.sub.4 together with the carbon to which they are
bound represent C.dbd.O, C.dbd.S, or C.dbd.NR.sub.8 g; R.sub.5 is
polyheteroaryl which is substituted or unsubstituted indol-3-yl; n,
n.sub.1, n.sub.2 and n.sub.3 are the same or different and
independently selected from 0-6, when n, is 1-6, each carbon atom
can be optionally and independently substituted with R.sub.3 and/or
R.sub.4 ; X and Y are the same or different and independently
selected from H, halo, C.sub.1 -C.sub.4 alkyl, NO.sub.2,
C(O)R.sub.1, OR.sub.9, SR.sub.9, CN, and NR.sub.10 R.sub.11 ;
R.sub.6 is selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl,
cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,
OR.sub.12, and NR.sub.13 R.sub.14 ; R.sub.7 is selected from
OR.sub.15, SR.sub.15, S(O)R.sub.16, SO.sub.2 R.sub.17, NR.sub.13
R.sub.14, and NR.sub.12 SO.sub.2 R.sub.6 ; R.sub.8 is selected from
H, OR.sub.15, NR.sub.13 R.sub.14, C.sub.1 -C.sub.6 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl,
heteroaryl, arylalkyl, and heteroarylalkyl; R.sub.9 is selected
from C.sub.1 -C.sub.4 alkyl and C(O)-alkyl; R.sub.10 and R.sub.11
are the same or different and independently selected from H,
C.sub.1 -C.sub.4 alkyl, and --C(O)-alkyl; R.sub.12 is selected from
H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4
-C.sub.9 heterocycloalkyl, C.sub.4 -C.sub.9 heterocycloalkylalkyl,
aryl, mixed aryl and non-aryl polycycle, heteroaryl, arylalkyl, and
heteroarylalkyl; R.sub.13 and R.sub.14 are the same or different
and independently selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, amino acyl, or R.sub.13 and
R.sub.14 together with the nitrogen to which they are bound are
C.sub.4 -C.sub.9 heterocycloalkyl, heteroaryl, polyheteroaryl,
non-aromatic polyheterocycle or mixed aryl and non-aryl
polyheterocycle; R.sub.15 is selected from H, C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and
(CH.sub.2).sub.m ZR.sub.12 ; R.sub.16 is selected from C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, polyheteroaryl, arylalkyl,
heteroarylalkyl and (CH.sub.2).sub.m ZR.sub.12 ; R.sub.17 is
selected from C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl,
C.sub.4 -C.sub.9 heterocycloalkyl, aryl, aromatic polycycle,
heteroaryl, arylalkyl, heteroarylalkyl, polyheteroaryl and
NR.sub.13 R.sub.14 ; m is an integer selected from 0 to 6; and Z is
selected from O, NR.sub.13, S and S(O);
or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier.
22. A pharmaceutical composition of claim 21 wherein the compound
of formula (I) is selected from the group consisting of
N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phe
nyl]-2E-2-propenamide, or a pharmaceutically acceptable salt
thereof.
23. A compound of claim 1 wherein R.sub.3 and R.sub.4 are each
H.
24. A compound of claim 8, wherein R.sub.1, R.sub.3 and R.sub.4 are
each H, X and Y are each H, R.sub.20 is H or C.sub.1 -C.sub.4 alkyl
and p is 1.
25. A pharmaceutical composition of claim 21, which comprises a
pharmaceutically effective amount of a compound of formula (Ic)
##STR315##
wherein Z.sub.1 is N--R.sub.20 ; R.sub.18 is H, halo, C.sub.1
-C.sub.6 alkyl, C.sub.3 -C.sub.7 cycloalkyl, aryl, or heteroaryl;
R.sub.20 is H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6
alkyl-C.sub.3 -C.sub.9 cycloalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, acyl or sulfonyl; A.sub.1 is 1, 2 or 3
substituents which are independently H, C.sub.1 -C-.sub.6 alkyl,
--OR.sub.19, halo, alkylamino, aminoalkyl, halo, or
heteroarylalkyl, R.sub.2 is selected from H, C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, --(CH.sub.2).sub.n C(O)R.sub.6, amino acyl and
--(CH.sub.2).sub.n R.sub.7 ; R.sub.19 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;
p is 0-3, and q is 1-5 and r is 0 or q is 0 and r is 1-5
or a pharmaceutically acceptable salt thereof.
26. A pharmaceutical composition of claim 25, which comprises a
pharmaceutically effective amount a compound of formula (Ic)
wherein R.sub.18 is H, halo, C.sub.1 -C.sub.6 alkyl, C.sub.3
-C.sub.7 cycloalkyl, unsubstituted phenyl, substituted phenyl, or
heteroaryl, R.sub.20 is H, C.sub.1 -C.sub.6 alkyl-C.sub.1 -C.sub.6
alkyl-C.sub.3 -C.sub.9 cycloalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, acyl or sulfonyl; A.sub.1 is 1, 2 or 3
substituents which are independently H, C.sub.1 -C.sub.6 alkyl,
--OR.sub.19, or halo, or a pharmaceutically acceptable salt
thereof.
27. A pharmaceutical composition of claim 25, wherein R.sub.1,
R.sub.3 and R.sub.4 are each H, R.sub.2 is H or --CH.sub.2 CH.sub.2
OH, X and Y are each H, R.sub.20 is H or C.sub.1 -C.sub.4 alkyl, p
is 1 and q is 1.
Description
The present invention relates to hydroxamate compounds which are
inhibitors of histone deacetylase. The inventive compounds are
useful as pharmaceuticals for the treatment of proliferative
diseases.
BACKGROUND
Reversible acetylation of histones is a major regulator of gene
expression that acts by altering accessibility of transcription
factors to DNA. In normal cells, histone deacetylase (HDA) and
histone acetyltrasferase together control the level of acetylation
of histones to maintain a balance. Inhibition of HDA results in the
accumulation of hyperacetylated histones, which results in a
variety of cellular responses.
Inhibitors of HDA have been studied for their therapeutic effects
on cancer cells. For example, butyric acid and its derivatives,
including sodium phenylbutyrate, have been reported to induce
apoptosis in vitro in human colon carcinoma, leukemia and
retinoblastoma cell lines. However, butyric acid and its
derivatives are not useful pharmacological agents because they tend
to be metabolized rapidly and have a very short half-life in vivo.
Other inhibitors of HDA that have been widely studied for their
anti-cancer activities are trichostatin A and trapoxin.
Trichostatin A is an antifungal and antibiotic and is a reversible
inhibitor of mammalian HDA Trapoxin is a cyclic tetrapeptide, which
is an irreversible inhibitor of mammalian HDA. Although
trichostatin and trapoxin have been studied for their anti-cancer
activities, the in vivo instability of the compounds makes them
less suitable as anti-cancer drugs. There remains a need for an
active compound that is suitable for treating tumors, including
cancerous tumors, that is highly efficacious and stable.
SUMMARY
The present invention provides efficacious deacetylase inhibitor
compounds that are useful as pharmaceutical agents having the
formula (I): ##STR1##
wherein R.sub.1 is H, halo, or a straight chain C.sub.1 -C.sub.6
alkyl (especially methyl, ethyl or n-propyl, which methyl, ethyl
and n-propyl substituents are unsubstituted or substituted by one
or more substituents described below for alkyl substituents);
R.sub.2 is selected from H, C.sub.1 -C.sub.10 alkyl, (e.g. methyl,
ethyl or --CH.sub.2 CH.sub.2 --OH), C.sub.4 -C.sub.9 cycloalkyl,
C.sub.4 -C.sub.9 heterocycloalkyl, C.sub.4 -C.sub.9
heterocycloalkylalkyl, cycloalkylalkyl (e.g., cyclopropylmethyl),
aryl, heteroaryl, arylalkyl (e.g. benzyl), heteroarylalkyl (e.g.
pyridylmethyl), --(CH.sub.2).sub.n C(O)R.sub.6, --(CH.sub.2).sub.n
OC(O)R.sub.6, amino acyl, HON--C(O)--CH.dbd.C(R.sub.1)-aryl-alkyl-
and --(CH.sub.2).sub.n R.sub.7 ; R.sub.3 and R.sub.4 are the same
or different and independently H, C.sub.1 -C.sub.6 alkyl, acyl or
acylamino, or R.sub.3 and R.sub.4 together with the carbon to which
they are bound represent C.dbd.O, C.dbd.S, or C.dbd.NR.sub.8, or
R.sub.2 together with the nitrogen to which it is bound and R.sub.3
together with the carbon to which it is bound can form a C.sub.4
-C.sub.9 heterocycloalkyl, a heteroaryl, a polyheteroaryl, a
non-aromatic polyheterocycle, or a mixed aryl and non-aryl
polyheterocycle ring; R.sub.5 is selected from H, C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, acyl, aryl, heteroaryl, arylalkyl (e.g. benzyl),
heteroarylalkyl (e.g. pyridylmethyl), aromatic polycycles,
non-aromatic polycycles, mixed aryl and non-aryl polycycles,
polyheteroaryl, non-aromatic polyheterocycles, and mixed aryl and
non-aryl polyheterocycles; n, n.sub.1, n.sub.2 and n.sub.3 are the
same or different and independently selected from 0-6, when n.sub.1
is 1-6, each carbon atom can be optionally and independently
substituted with R.sub.3 and/or R.sub.4 ; X and Y are the same or
different and independently selected from H, halo, C.sub.1 -C.sub.4
alkyl, such as CH.sub.3 and CF.sub.3, NO.sub.2, C(O)R.sub.1,
OR.sub.9, SR.sub.9, CN, and NR.sub.10 R.sub.11 ; R.sub.6 is
selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9
cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, cycloalkylalkyl
(e.g., cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g.,
benzyl, 2-phenylethenyl), heteroarylalkyl (e.g., pyridylmethyl),
OR.sub.12, and NR.sub.13 R.sub.14 ; R.sub.7 is selected from
OR.sub.15, SR.sub.15, S(O)R.sub.16, SO.sub.2 R.sub.17, NR.sub.13
R.sub.14, and NR.sub.12 SO.sub.2 R.sub.6 ; R.sub.8 is selected from
H, OR.sub.15, NR.sub.13 R.sub.14, C.sub.1 -C.sub.6 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl,
heteroaryl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g.,
pyridylmethyl); R.sub.9 is selected from C.sub.1 -C.sub.4 alkyl,
for example, CH.sub.3 and CF.sub.3, C(O)-alkyl, for example
C(O)CH.sub.3, and C(O)CF.sub.3 ; R.sub.10 and R.sub.11 are the same
or different and independently selected from H, C.sub.1 -C.sub.4
alkyl, and --C(O)-alkyl; R.sub.12 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, C.sub.4 -C.sub.9 heterocycloalkylalkyl, aryl,
mixed aryl and non-aryl polycycle, heteroaryl, arylalkyl (e.g.,
benzyl), and heteroarylalkyl (e.g., pyridylmethyl); R.sub.13 and
R.sub.14 are the same or different and independently selected from
H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4
-C.sub.9 heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g.,
benzyl), heteroarylalkyl (e.g., pyridylmethyl), amino acyl, or
R.sub.13 and R.sub.14 together with the nitrogen to which they are
bound are C.sub.4 -C.sub.9 heterocycloalkyl, heteroaryl,
polyheteroaryl, non-aromatic polyheterocycle or mixed aryl and
non-aryl polyheterocycle; R.sub.15 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and
(CH.sub.2).sub.m ZR.sub.12 ; R.sub.16 is selected from C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, polyheteroaryl, arylalkyl,
heteroarylalkyl and (CH.sub.2).sub.m ZR.sub.12 ; R.sub.17 is
selected from C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl,
C.sub.4 -C.sub.9 heterocycloalkyl, aryl, aromatic polycycles,
heteroaryl, arylalkyl, heteroarylalkyl, polyheteroaryl and
NR.sub.13 R.sub.14 ; m is an integer selected from 0 to 6; and Z is
selected from O NR.sub.13, S and S(O),
or a pharmaceutically acceptable salt thereof.
The compounds of the present invention are suitable as active
agents in pharmaceutical compositions that are efficacious
particularly for treating cellular proliferative ailments. The
pharmaceutical composition has ia pharmaceutically effective amount
of the present active agent along with other pharmaceutically
acceptable exicipients, carriers, fillers, diluents and the like.
The term pharmacuetically effective amount as used herein indicates
an amount necessary to administer to a host to achieve a
therapeutic result, especially an anti-tumor effect, e.g.,
inhibition of proliferation of malignant cancer cells, benign tumor
cells or other proliferative cells.
DETAILED DESCRIPTION
The present invention provides hydroxamate compounds, e.g.,
hydroxamic acids, that are inhibitors of deacetylases, preferably
inhibitors of histone deacetylases. The hydroxamate compounds are
highly suitable for treating tumors, including cancerous tumors.
The hydroxamate compounds of the present invention have the
following structure (I): ##STR2##
wherein R.sub.1 is H, halo, or a straight chain C.sub.1 -C.sub.6
alkyl (especially methyl, ethyl or n-propyl, which methyl, ethyl
and n-propyl substituents are unsubstituted or substituted by one
or more substituents described below for alkyl substituents);
R.sub.2 is selected from H, C.sub.1 -C.sub.10 alkyl, (preferably
C.sub.1 -C.sub.6 alkyl, e.g. methyl, ethyl or --CH.sub.2 CH.sub.2
--OH), C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, C.sub.4 -C.sub.9 heterocycloalkylalkyl,
cycloalkylalkyl (e.g., cyclopropylmethyl), aryl, heteroaryl,
arylalkyl (e.g. benzyl), heteroarylalkyl (e.g. pyridylmethyl),
--(CH.sub.2).sub.n C(O)R.sub.6, --(CH.sub.2).sub.n OC(O)R.sub.6,
amino acyl, HON--C(O)--CH.dbd.C(R.sub.1)-aryl-alkyl- and
--(CH.sub.2).sub.n R.sub.7 ; R.sub.3 and R.sub.4 are the same or
different and independently H, C.sub.1 -C.sub.6 alkyl, acyl or
acylamino, or R.sub.3 and R.sub.4 together with the carbon to which
they are bound represent C.dbd.O, C.dbd.S, or C.dbd.NR.sub.8, or
R.sub.2 together with the nitrogen to which it is bound and R.sub.3
together with the carbon to which it is bound can form a C.sub.4
-C.sub.9 heterocycloalkyl, a heteroaryl, a polyheteroaryl, a
non-aromatic polyheterocycle, or a mixed aryl and non-aryl
polyheterocycle ring; R.sub.5 is selected from H, C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, acyl, aryl, heteroaryl, arylalkyl (e.g. benzyl),
heteroarylalkyl (e.g. pyridylmethyl), aromatic polycycles,
non-aromatic polycycles, mixed aryl and non-aryl polycycles,
polyheteroaryl, non-aromatic polyheterocycles, and mixed aryl and
non-aryl polyheterocycles; n, n.sub.1, n.sub.2 and n.sub.3 are the
same or different and independently selected from 0-6, when n.sub.1
is 1-6, each carbon atom can be optionally and independently
substituted with R.sub.3 and/or R.sub.4 ; X and Y are the same or
different and independently selected from H, halo, C.sub.1 -C.sub.4
alkyl, such as CH.sub.3 and CF.sub.3, NO.sub.2, C(O)R.sub.1,
OR.sub.9, SR.sub.9, CN, and NR.sub.10 R.sub.11 ; R.sub.6 is
selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9
cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, cycloalkylalkyl
(e.g., cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g.,
benzyl, 2-phenylethenyl), heteroarylalkyl (e.g., pyridylmethyl),
OR.sub.12, and NR.sub.13 R.sub.14 ; R.sub.7 is selected from
OR.sub.15, SR.sub.15, S(O)R.sub.16, SO.sub.2 R.sub.17, NR.sub.13
R.sub.14, and NR.sub.12 SO.sub.2 R.sub.6 ; R.sub.8 is selected from
H, OR.sub.15, NR.sub.13 R.sub.14, C.sub.1 -C.sub.6 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl,
heteroaryl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g.,
pyridylmethyl); R.sub.9 is selected from C.sub.1 -C.sub.4 alkyl,
for example, CH.sub.3 and CF.sub.3, C(O)-alkyl, for example
C(O)CH.sub.3, and C(O)CF.sub.3 ; R.sub.10 and R.sub.11 are the same
or different and independently selected from H, C.sub.1 -C.sub.4
alkyl, and --C(O)-alkyl; R.sub.12 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, C.sub.4 -C.sub.9 heterocycloalkylalkyl, aryl,
mixed aryl and non-aryl polycycle, heteroaryl, arylalkyl (e.g.,
benzyl), and heteroarylalkyl (e.g., pyridylmethyl); R.sub.13 and
R.sub.14 are the same or different and independently selected from
H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4
-C.sub.9 heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g.,
benzyl), heteroarylalkyl (e.g., pyridylmethyl), amino acyl, or
R.sub.13 and R.sub.14 together with the nitrogen to which they are
bound are C.sub.4 -C.sub.9 heterocycloalkyl, heteroaryl,
polyheteroaryl, non-aromatic polyheterocycle or mixed aryl and
non-aryl polyheterocycle; R.sub.15 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and
(CH.sub.2).sub.m ZR.sub.12 ; R.sub.16 is selected from C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, polyheteroaryl, arylalkyl,
heteroarylalkyl and (CH.sub.2).sub.m ZR.sub.12 ; R.sub.17 is
selected from C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl,
C.sub.4 -C.sub.9 heterocycloalkyl, aryl, aromatic polycycles,
heteroaryl, arylalkyl, heteroarylalkyl, polyheteroaryl and
NR.sub.13 R.sub.14 ; m is an integer selected from 0 to 6; and Z is
selected from O NR.sub.13, S and S(O),
or a pharmaceutically acceptable salt thereof.
As appropriate, unsubstituted means that there is no substituent or
that the only substituents are hydrogen.
Halo substituents are selected from fluoro, chloro, bromo and iodo,
preferably fluoro or chloro.
Alkyl substituents include straight and branched C.sub.1 -C.sub.6
alkyl, unless otherwise noted. Examples of suitable straight and
branched C.sub.1 -C.sub.6 alkyl substituents include methyl, ethyl,
n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, and the like.
Unless otherwise noted, the alkyl substituents include both
unsubstituted alkyl groups and alkyl groups that are substituted by
one or more suitable substituents, including unsaturation (i.e.
there are one or more double or triple C--C bonds), acyl,
cycloalkyl, halo, oxyalkyl, alkylamino, aminoalkyl, acylamino and
OR.sub.15, for example, alkoxy. Preferred substituents for alkyl
groups include halo, hydroxy, alkoxy, oxyalkyl, alkylamino, and
aminoalkyl.
Cycloalkyl substituents include C.sub.3 -C.sub.9 cycloalkyl groups,
such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the
like, unless otherwise specified. Unless otherwise noted,
cycloalkyl substituents include both unsubstituted cycloalkyl
groups and cycloalkyl groups that are substituted by one or more
suitable substituents, including C.sub.1 -C.sub.6 alkyl, halo,
hydroxy, aminoalkyl, oxyalkyl, alkylamino, and OR.sub.15, such as
alkoxy. Preferred substituents for cycloalkyl groups include halo,
hydroxy, alkoxy, oxyalkyl, alkylamino and aminoalkyl.
The above discussion of alkyl and cycloalkyl substituents also
applies to the alkyl portions of other substituents, such as
without limitation, alkoxy, alkyl amines, alkyl ketones, arylalkyl,
heteroarylalkyl, alkylsulfonyl and alkyl ester substituents and the
like.
Heterocycloalkyl substituents include 3 to 9 membered aliphatic
rings, such as 4 to 7 membered aliphatic rings, containing from one
to three heteroatoms selected from nitrogen, sulfur, oxygen.
Examples of suitable heterocycloalkyl substituents include
pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl,
piperazyl, tetrahydropyranyl, morphilino, 1,3-diazapane,
1,4-diazapane, 1,4-oxazepane, and 1,4-oxathiapane. Unless otherwise
noted, the rings are unsubstituted or substuted on the carbon atoms
by one or more suitable substituents, including C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, aryl, heteroaryl, arylalkyl
(e.g., benzyl), and heteroarylalkyl (e.g., pyridylmethyl), halo,
amino, alkyl amino and OR.sub.15, for example alkoxy. Unless
otherwise noted, nitrogen heteroatoms are unsubstituted or
substituted by H, C.sub.1 -C.sub.4 alkyl, arylalkyl (e.g., benzyl),
and heteroarylalkyl (e.g., pyridylmethyl), acyl, aminoacyl,
alkylsulfonyl, and arylsulfonyl.
Cycloalkylalkyl substituents include compounds of the formula
--CH.sub.2).sub.n5 -cycloalkyl wherein n5 is a number from 1-6.
Suitable alkylcycloalkyl substituents include cyclopentylmethyl-,
cyclopentylethyl, cyclohexylmethyl and the like. Such substituents
are unsubstituted or substituted in the alkyl portion or in the
cycloalkyl portion by a suitable substituent, including those
listed above for alkyl and cycloalkyl.
Aryl substituents include unsubstituted phenyl and phenyl
substituted by one or more suitable substituents, including C.sub.1
-C.sub.6 alkyl, cycloalkylalkyl (e.g., cyclopropylmethyl),
O(CO)alkyl, oxyalkyl, halo, nitro, amino, alkylamino, aminoalkyl,
alkyl ketones, nitrile, carboxyalkyl, alkylsulfonyl, aminosulfonyl,
arylsulfonyl, and OR.sub.15, such as alkoxy. Preferred substituents
include including C.sub.1 -C.sub.6 alkyl, cycloalkyl (e.g.,
cyclopropylmethyl), alkoxy, oxyalkyl, halo, nitro, amino,
alkylamino, aminoalkyl, alkyl ketones, nitrile, carboxyalkyl,
alkylsulfonyl, arylsulfonyl, and aminosulfonyl. Examples of
suitable aryl groups include C.sub.1 -C.sub.4 alkylphenyl, C.sub.1
-C.sub.4 alkoxyphenyl, trifluoromethylphenyl, methoxyphenyl,
hydroxyethylphenyl, dimethylaminophenyl, aminopropylphenyl,
carbethoxyphenyl, methanesulfonylphenyl and
tolylsulfonylphenyl.
Aromatic polycycles include naphthyl, and naphthyl substituted by
one or more suitable substituents, including C.sub.1 -C.sub.6
alkyl, alkylcycloalkyl (e.g., cyclopropylmethyl), oxyalkyl, halo,
nitro, amino, alkylamino, aminoalkyl, alkyl ketones, nitrile,
carboxyalkyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl and
OR.sub.15, such as alkoxy.
Heteroaryl substituents include compounds with a 5 to 7 member
aromatic ring containing one or more heteroatoms, for example from
1 to 4 heteroatoms, selected from N, O and S. Typical heteroaryl
substituents include furyl, thienyl, pyrrole, pyrazole, triazole,
thiazole, oxazole, pyridine, pyrimidine, isoxazolyl, pyrazine and
the like. Unless otherwise noted, heteroaryl substituents are
unsubstituted or substituted on a carbon atom by one or more
suitable substituents, including alkyl, the alkyl substituents
identified above, and another heteroaryl substituent. Nitrogen
atoms are unsubstituted or substituted, for example by R.sub.13 ;
especially useful N substituents include H, C.sub.1 -C.sub.4 alkyl,
acyl, aminoacyl, and sulfonyl.
Arylalkyl substituents include groups of the formula
--(CH.sub.2).sub.n5 -aryl, --(CH.sub.2).sub.n5-1
--(CHaryl)--(CH.sub.2).sub.n5 -aryl or --(CH.sub.2).sub.n5-1
CH(aryl)(aryl) wherein aryl and n5 are defined above. Such
arylalkyl substituents include benzyl, 2-phenylethyl,
1-phenylethyl, tolyl-3-propyl, 2-phenylpropyl, diphenylmethyl,
2-diphenylethyl, 5,5-dimethyl-3-phenylpentyl and the like.
Arylalkyl substituents are unsubstituted or substituted in the
alkyl moiety or the aryl moiety or both as described above for
alkyl and aryl substituents.
Heteroarylalkyl substituents include groups of the formula
--(CH.sub.2).sub.n5 -heteroaryl wherein heteroaryl and n5 are
defined above and the bridging group is linked to a carbon or a
nitrogen of the heteroaryl portion, such as 2- , 3- or
4-pyridylmethyl, imidazolylmethyl, quinolylethyl, and
pyrrolylbutyl. Heteroaryl substituents are unsubstituted or
substituted as discussed above for heteroaryl and alkyl
substituents.
Amino acyl substituents include groups of the formula
--C(O)--(CH.sub.2).sub.n --C(H)(NR.sub.13
R.sub.14)--(CH.sub.2).sub.n --R.sub.5 wherein n, R.sub.13, R.sub.14
and R.sub.5 are described above. Suitable aminoacyl substituents
include natural and non-natural amino acids such as glycinyl,
D-tryptophanyl, L-lysinyl, D- or L-homoserinyl, 4-aminobutryic
acyl, .+-.-3-amin-4-hexenoyl.
Non-aromatic polycycle substituents include bicyclic and tricyclic
fused ring systems where each ring can be 4-9 membered and each
ring can contain zero, 1 or more double and/or triple bonds.
Suitable examples of non-aromatic polycycles include decalin,
octahydroindene, perhydrobenzocycloheptene,
perhydrobenzo-[f]-azulene. Such substituents are unsubstituted or
substituted as described above for cycloalkyl groups.
Mixed aryl and non-aryl polycycle substituents include bicyclic and
tricyclic fused ring systems where each ring can be 4-9 membered
and at least one ring is aromatic. Suitable examples of mixed aryl
and non-aryl polycycles include methylenedioxyphenyl,
bis-methylenedioxyphenyl, 1,2,3,4-tetrahydronaphthalene,
dibenzosuberane, dihdydroanthracene, 9H-fluorene. fluorene. Such
substituents are unsubstituted or substituted by nitro or as
described above for cycloalkyl groups.
Polyheteroaryl substituents include bicyclic and tricyclic fused
ring systems where each ring can independently be 5 or 6 membered
and contain one or more heteroatom, for example, 1, 2, 3, or 4
heteroatoms, chosen from O, N or S such that the fused ring system
is aromatic. Suitable examples of polyheteroaryl ring systems
include quinoline, isoquinoline, pyridopyrazine, pyrrolopyridine,
furopyridine, indole, benzofuran, benzothiofuran, benzindole,
benzoxazole, pyrroloquinoline, and the like. Unless otherwise
noted, polyheteroaryl substituents are unsubstituted or substituted
on a carbon atom by one or more suitable substituents, including
alkyl, the alkyl substituents identified above and a substituent of
the formula --O--(CH.sub.2 CH.dbd.CH(CH.sub.3)(CH.sub.2)).sub.1-3
H. Nitrogen atoms are unsubstituted or substituted, for example by
R.sub.13 ; especially useful N substituents include H, C.sub.1
-C.sub.4 alkyl, acyl, aminoacyl, and sulfonyl.
Non-aromatic polyheterocyclic substituents include bicyclic and
tricyclic fused ring systems where each ring can be 4-9 membered,
contain one or more heteroatom, for example, 1, 2, 3, or 4
heteroatoms, chosen from O, N or S and contain zero or one or more
C--C double or triple bonds. Suitable examples of non-aromatic
polyheterocycles include hexitol,
cis-perhydro-cyclohepta[b]pyridinyl,
decahydro-benzo[f][1,4]oxazepinyl, 2,8-dioxabicyclo[3.30]octane,
hexahydro-thieno[3,2-b]thiophene, perhydropyrrolo[3,2-b]pyrrole,
perhydronaphthyridine, perhydro-1H-dicyclopenta[b,e]pyran. Unless
otherwise noted, non-aromatic polyheterocyclic substituents are
unsubstituted or substituted on a carbon atom by one or more
substituents, including alkyl and the alkyl substituents identified
above. Nitrogen atoms are unsubstituted or substituted, for
example, by R.sub.13 ; especially useful N substituents include H,
C.sub.1 -C.sub.4 alkyl, acyl, aminoacyl, and sulfonyl.
Mixed aryl and non-aryl polyheterocycles substituents include
bicyclic and tricyclic fused ring systems where each ring can be
4-9 membered, contain one or more heteroatom chosen from O, N or S,
and at least one of the rings must be aromatic. Suitable examples
of mixed aryl and non-aryl polyheterocycles include
2,3-dihydroindole, 1,2,3,4-tetrahydroquinoline,
5,11-dihydro-10H-dibenz[b,e][1,4]diazepine,
5H-dibenzo[b,e][1,4]diazepine,
1,2-dihydropyrrolo[3,4-b][1,5]benzodiazepine,
1,5-dihydro-pyrido[2,3-b][1,4]diazepin-4-one,
1,2,3,4,6,11-hexahydro-benzo[b]pyrido[2,3-e][1,4]diazepin-5-one.
Unless otherwise noted, mixed aryl and non-aryl polyheterocyclic
substituents are unsubstituted or substituted on a carbon atom by
one or more suitable substituents, including, --N--OH, .dbd.N--OH,
alkyl and the alkyl substituents identified above. Nitrogen atoms
are unsubstituted or substituted, for example, by R.sub.13 ;
especially useful N substituents include H, C.sub.1 -C.sub.4 alkyl,
acyl, aminoacyl, and sulfonyl.
Amino substituents include primary, secondary and tertiary amines
and in salt form, quatemary amines. Examples of amino substituents
include mono- and di-alkylamino, mono- and di-aryl amino, mono- and
di-arylalkyl amino, aryl-arylalkylamino, alkyl-arylamino,
alkyl-arylalkylamino and the like.
Sulfonyl substituents include alkylsulfonyl and arylsulfonyl, for
example methane sulfonyl, benzene sulfonyl, tosyl and the like.
Acyl substituents include groups of formula --C(O)--W, --OC(O)--W,
--C(O)--O--W or --C(O)NR.sub.13 R.sub.14, where W is R.sub.16, H or
cycloalkylalkyl.
Acylamino substituents include substituents of the formula
--N(R.sub.12)C(O)--W, --N(R.sub.12)C(O)--O--W, and
--N(R.sub.12)C(O)--NHOH and R.sub.12 and W are defined above.
The R.sub.2 substituent HON--C(O)CH.dbd.C(R.sub.1)-aryl-alkyl- is a
group of the formula ##STR3##
Preferences for each of the substituents include the following:
R.sub.1 is H, halo, or a straight chain C.sub.1 -C.sub.4 alkyl;
R.sub.2 is selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4
-C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl,
alkylcycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,
--(CH.sub.2).sub.n C(O)R.sub.6, amino acyl, and --(CH.sub.2).sub.n
R.sub.7 ; R.sub.3 and R.sub.4 are the same or different and
independently selected from H, and C.sub.1 -C.sub.6 alkyl, or
R.sub.3 and R.sub.4 together with the carbon to which they are
bound represent C.dbd.O, C.dbd.S, or C.dbd.NR.sub.8 ; R.sub.5 is
selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9
cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl, heteroaryl,
arylalkyl, heteroarylalkyl, a aromatic polycycle, a non-aromatic
polycycle, a mixed aryl and non-aryl polycycle, polyheteroaryl, a
non-aromatic polyheterocycle, and a mixed aryl and non-aryl
polyheterocycle; n, n.sub.1, n.sub.2 and n.sub.3 are the same or
different and independently selected from 0-6, when n.sub.1 is 1-6,
each carbon atom is unsubstituted or independently substituted with
R.sub.3 and/or R.sub.4 ; X and Y are the same or different and
independently selected from H, halo, C.sub.1 -C.sub.4 alkyl,
CF.sub.3, NO.sub.2, C(O)R.sub.1, OR.sub.9, SR.sub.9, CN, and
NR.sub.10 R.sub.11 ; R.sub.6 is selected from H, C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, OR.sub.12, and NR.sub.13 R.sub.14 ; R.sub.7 is
selected from OR.sub.15, SR.sub.15, S(O)R.sub.16, SO.sub.2
R.sub.17, NR.sub.13 R.sub.14, and NR.sub.12 SO.sub.2 R.sub.6 ;
R.sub.8 is selected from H, OR.sub.15, NR.sub.13 R.sub.14, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;
R.sub.9 is selected from C.sub.1 -C.sub.4 alkyl and C(O)-alkyl;
R.sub.10 and R.sub.11 are the same or different and independently
selected from H, C.sub.1 -C.sub.4 alkyl, and --C(O)-alkyl; R.sub.12
is selected from H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9
cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl, aryl, heteroaryl,
arylalkyl, and heteroarylalkyl; R.sub.13 and R.sub.14 are the same
or different and independently selected from H, C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and
amino acyl; R.sub.15 is selected from H, C.sub.1 -C.sub.6 alkyl,
C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl,
aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH.sub.2).sub.m
ZR.sub.12 ; R.sub.16 is selected from C.sub.1 -C.sub.6 alkyl,
C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocycloalkyl,
aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH.sub.2).sub.m
ZR.sub.12 ; R.sub.17 is selected from C.sub.1 -C.sub.6 alkyl,
C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9 heterocydoalkyl,
aryl, heteroaryl, arylalkyl, heteroarylalkyl and NR.sub.13 R.sub.14
; m is an integer selected from 0 to 6; and Z is selected from O,
NR.sub.13, S, S(O),
or a pharmaceutically acceptable salt thereof.
Useful compounds of the formula (I) include those wherein each of
R.sub.1, X, Y, R.sub.3, and R.sub.4 is H, including those wherein
one of n.sub.2 and n.sub.3 is zero and the other is 1, especially
those wherein R.sub.2 is H or --CH.sub.2 --CH.sub.2 --OH.
One suitable genus of hydroxamate compounds are those of formula
Ia: ##STR4##
wherein n.sub.4 is 0-3, R.sub.2 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, --(CH.sub.2).sub.n C(O)R.sub.6, amino acyl and
--(CH.sub.2).sub.n R.sub.7 ; R.sub.5 ' is heteroaryl,
heteroarylalkyl (e.g., pyridylmethyl), aromatic polycycles,
non-aromatic polycycles, mixed aryl and non-aryl polycycles,
polyheteroaryl, or mixed aryl and non-aryl polyheterocycles,
or a pharmaceutically acceptable salt thereof.
Another suitable genus of hydroxamate compounds are those of
formula Ia: ##STR5##
wherein n.sub.4 is 0-3, R.sub.2 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, --(CH.sub.2).sub.n C(O)R.sub.6, amino acyl and
--(CH.sub.2).sub.n R.sub.7 ; R.sub.5 ' is aryl, arylalkyl, aromatic
polycycles, non-aromatic polycycles, and mixed aryl and non-aryl
polycycles; especially aryl, such as p-fluorophenyl,
p-chlorophenyl, p-O-C.sub.1 -C.sub.4 -alkylphenyl, such as
p-methoxyphenyl, and p-C.sub.1 -C.sub.4 -alkylphenyl; and
arylalkyl, such as benzyl, ortho, meta or para-fluorobenzyl, ortho,
meta or para-chlorobenzyl, ortho, meta or para-mono, di or
tri-O-C.sub.1 -C.sub.4 -alkylbenzyl, such as ortho, meta or
para-methoxybenzyl, m,p-diethoxybenzyl, o,m,p-triimethoxybenzyl ,
and ortho, meta or para- mono, di or tri C.sub.1 -C.sub.4
-alkylphenyl, such as p-methyl, m,m-diethylphenyl,
or a pharmaceutically acceptable salt thereof.
Another interesting genus are the compounds of formula Ib:
##STR6##
wherein R.sub.2 ' is selected from H, C.sub.1 -C.sub.6 alkyl,
C.sub.4 -C.sub.6 cycloalkyl, cycloalkylalkyl (e.g.,
cyclopropylmethyl), (CH.sub.2).sub.2-4 OR.sub.21 where R.sub.21 is
H, methyl, ethyl, propyl, and i-propyl, and R.sub.5 " is
unsubstituted 1H-indol-3-yl, benzofuran-3-yl or quinolin-3-yl, or
substituted 1H-indol-3-yl, such as 5-fluoro-1H-indol-3-yl or
5-methoxy-1H-indol-3-yl, benzofuran-3-yl or quinolin-3-yl,
or a pharmaceutically acceptable salt thereof.
Another interesting genus of hydroxamate compounds are the
compounds of formula (Ic) ##STR7##
wherein the ring containing Z.sub.1 is aromatic or non-aromatic,
which non-aromatic rings are saturated or unsaturated, Z.sub.1 is
O, S or N--R.sub.20, R18 is H, halo, C.sub.1 -C.sub.6 alkyl
(methyl, ethyl, t-butyl), C.sub.3 -C.sub.7 cycloalkyl, aryl, for
example unsubstituted phenyl or phenyl substituted by 4-OCH.sub.3
or 4-CF.sub.3, or heteroaryl, such as 2-furanyl, 2-thiophenyl or
2-, 3- or 4-pyridyl; R.sub.20 is H, C.sub.1 -C.sub.6 alkyl, C.sub.1
-C.sub.6 alkyl-C.sub.3 -C.sub.9 cycloalkyl (e.g.,
cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g., benzyl),
heteroarylalkyl (e.g., pyridylmethyl), acyl (acetyl, propionyl,
benzoyl) or sulfonyl (methanesulfonyl, ethanesulfonyl,
benzenesulfonyl, toluenesulfonyl) A.sub.1 is 1, 2 or 3 substituents
which are independently H, C.sub.1 -C-.sub.6 alkyl, --OR.sub.9,
halo, alkylamino, aminoalkyl, halo, or heteroarylalkyl (e.g.,
pyridylmethyl), R.sub.19 is selected from H, C.sub.1 -C.sub.6
alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl),
heteroarylalkyl (e.g., pyridylmethyl) and --(CH.sub.2
CH.dbd.CH(CH.sub.3)(CH.sub.2)).sub.1-3 H; R.sub.2 is selected from
H, C.sub.1 -C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4
-C.sub.9 heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl,
arylalkyl, heteroarylalkyl, --(CH.sub.2).sub.n C(O)R.sub.6, amino
acyl and --(CH.sub.2).sub.n R.sub.7 ; v is 0, 1 or 2, p is 0-3, and
q is 1-5 and r is 0 or q is 0 and r is 1-5,
or a pharmaceutically acceptable salt thereof. The other variable
substituents are as defined above.
Especially useful compounds of formula (Ic) are those wherein
R.sub.2 is H, or --(CH.sub.2).sub.p CH.sub.2 OH, wherein p is 1-3,
especially those wherein R.sub.1 is H; such as those wherein
R.sub.1 is H and X and Y are each H, and wherein q is 1-3 and r is
0 or wherein q is 0 and r is 1-3, especially those wherein Z.sub.1
is N--R.sub.20. Among these compounds R.sub.2 is preferably H or
--CH.sub.2 --CH.sub.2 --OH and the sum of q and r is preferably
1.
Another interesting genus of hydroxamate compounds are the
compounds of formula (Id) ##STR8##
wherein Z.sub.1 is O, S or N--R.sub.20, R18 is H, halo, C.sub.1
-C.sub.6 alkyl (methyl, ethyl, t-butyl), C.sub.3 -C.sub.7
cycloalkyl, aryl, for example, unsubstituted phenyl or phenyl
substituted by 4-OCH.sub.3 or 4-CF.sub.3, or heteroaryl, R.sub.20
is H, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkyl-C.sub.3
-C.sub.9 cycloalkyl (e.g., cyclopropylmethyl), aryl, heteroaryl,
arylalkyl (e.g., benzyl), heteroarylalkyl (e.g., pyridylmethyl),
acyl (acetyl, propionyl, benzoyl) or sulfonyl (methanesulfonyl,
ethanesulfonyl, benzenesulfonyl, toluenesulfonyl), A.sub.1 is 1, 2
or 3 substituents which are independently H, C.sub.1 -C-.sub.6
alkyl, --OR.sub.19, or halo, R.sub.19 is selected from H, C.sub.1
-C.sub.6 alkyl, C.sub.4 -C.sub.9 cycloalkyl, C.sub.4 -C.sub.9
heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), and
heteroarylalkyl (e.g., pyridylmethyl); p is 0-3, and q is 1-5 and r
is 0 or q is 0 and r is 1-5,
or a pharmaceutically acceptable salt thereof. The other variable
substituents are as defined above.
Especially useful compounds of formula (Id) are those wherein
R.sub.2 is H, or --(CH.sub.2).sub.p CH.sub.2 OH, wherein p is 1-3,
especially those wherein R.sub.1 is H; such as those wherein
R.sub.1 is H and X and Y are each H, and wherein q is 1-3 and r is
0 or wherein q is 0 and r is 1-3. Among these compounds R.sub.2 is
preferably H or --CH.sub.2 --CH.sub.2 --OH and the sum of q and r
is preferably 1.
The present invention further relates to compounds of the formula
(Ie) ##STR9##
or a pharmaceutically acceptable salt thereof. The variable
substituents are as defined above.
Especially useful compounds of formula (Ie) are those wherein R8 is
H, fluoro, chloro, bromo, a C.sub.1 -C.sub.4 alkyl group, a
substituted C.sub.1 -C.sub.4 alkyl group, a C.sub.3 -C.sub.7
cycloalkyl group, unsubstituted phenyl, phenyl substituted in the
para position, or a heteroaryl (e.g., pyridyl) ring.
Another group of useful compounds of formula (Ie) are those wherein
R.sub.2 is H, or --(CH.sub.2).sub.p CH.sub.2 OH, wherein p is 1-3,
especially those wherein R.sub.1 is H; such as those wherein
R.sub.1 is H and X and Y are each H, and wherein q is 1-3 and r is
0 or wherein q is 0 and r is 1-3. Among these compounds R.sub.2 is
preferably H or --CH.sub.2 --CH.sub.2 OH and the sum of q and r is
preferably 1.
Another group of useful compounds of formula (le) are those wherein
R18 is H, methyl, ethyl, t-butyl, trifluoromethyl, cyclohexyl,
phenyl, 4-methoxyphenyl, 4-trifluoromethylphenyl, 2-furanyl,
2-thiophenyl, or 2-, 3- or 4-pyridyl wherein the 2-furanyl,
2-thiophenyl and 2-, 3or 4-pyridyl substituents are unsubstituted
or substituted as described above for heteroaryl rings; R.sub.2 is
H, or --(CH.sub.2).sub.p CH.sub.2 OH, wherein p is 1-3; especially
those wherein R.sub.1 is H and X and Y are each H, and wherein q is
1-3 and r is 0 or wherein q is 0 and r is 1-3. Among these
compounds R.sub.2 is preferably H or --CH.sub.2 --CH.sub.2 --OH and
the sum of q and r is preferably 1.
Those compounds of formula le wherein R.sub.20 is H or C.sub.1
-C.sub.6 alkyl, especially H, are important members of each of the
subgenuses of compounds of formula Ie described above.
N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phen
yl]-2E-2-propenamide,
N-hydroxy-3-[4-[[[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propen
amide and
N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2
E-2-propenamide, or a pharmaceutically acceptable salt thereof, are
important compounds of formula (Ie).
The present invention further relates to the compounds of the
formula (If): ##STR10##
or a pharmaceutically acceptable salt thereof. The variable
substituents are as defined above.
Useful compounds of formula (If) are include those wherein R.sub.2
is H, or --(CH.sub.2).sub.p CH.sub.2 OH, wherein p is 1-3,
especially those wherein R.sub.1 is H; such as those wherein
R.sub.1 is H and X and Y are X each H, and wherein q is 1-3 and r
is 0 or wherein q is 0 and r is 1-3. Among these compounds R.sub.2
is preferably H or --CH.sub.2 --CH.sub.2 --OH and the sum of q and
r is preferably 1.
N-hydroxy-3-[4-[[[2-(benzofur-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propen
amide, or a pharmaceutically acceptable salt thereof, is an
important compound of formula (If).
The compounds described above are often used in the form of a
pharmaceutically acceptable salt. Pharmaceutically acceptable salts
include, when appropriate, pharmaceutically acceptable base
addition salts and acid addition salts, for example, metal salts,
such as alkali and alkaline earth metal salts, ammonium salts,
organic amine addition salts, and amino acid addition salts, and
sulfonate salts. Acid addition salts include inorganic acid
addition salts such as hydrochloride, sulfate and phosphate, and
organic acid addition salts such as alkyl sulfonate, arylsulfonate,
acetate, maleate, fumarate, tartrate, citrate and lactate. Examples
of metal salts are alkali metal salts, such as lithium salt, sodium
salt and potassium salt, alkaline earth metal salts such as
magnesium salt and calcium salt, aluminum salt, and zinc salt.
Examples of ammonium salts are ammonium salt and
tetramethylammonium salt. Examples of organic amine addition salts
are salts with morpholine and piperidine. Examples of amino acid
addition salts are salts with glycine, phenylalanine, glutamic acid
and lysine. Sulfonate salts include mesylate, tosylate and benzene
sulfonic acid salts.
As is evident to those skilled in the art, the many of the
deacetylase inhibitor compounds of the present invention contain
asymmetric carbon atoms. It should be understood, therefore, that
the individual stereoisomers are contemplated as being included
within the scope of this invention.
The hydroxamate compounds of the present invention can be produced
by known organic synthesis methods. For example, the hydroxamate
compounds can be produced by reacting methyl 4-formyl cinnamate
with tryptamine and then converting the reactant to the hydroxamate
compounds. As an example, methyl 4-formyl cinnamate 2, is prepared
by acid catalyzed esterification of 4-formylcinnamic acid 3 (Bull.
Chem. Soc. Jpn. 1995; 68:2355-2362). An alternate preparation of
methyl 4-formyl cinnamate 2 is by a Pd-catalyzed coupling of methyl
acrylate 4 with 4-bromobenzaldehyde 5. ##STR11##
Additional starting materials can be prepared from
4-carboxybenzaldehyde 6, and an exemplary method is illustrated for
the preparation of aldehyde 9, shown below. The carboxylic acid in
4-carboxybenzaldehyde 6 can be protected as a silyl ester (e.g.,
the t-butyldimethylsilyl ester) by treatment with a silyl chloride
(e.g., t-butyldimethylsilyl chloride) and a base (e.g.
triethylamine) in an appropriate solvent (e.g., dichloromethane).
The resulting silyl ester 7 can undergo an olefination reaction
(e.g., a Homer-Emmons olefination) with a phosphonate ester (e.g.,
triethyl 2-phosphonopropionate) in the presence of a base (e.g.,
sodium hydride) in an appropriate solvent (e.g., tetrahydrofuran
(THF)). Treatment of the resulting diester with acid (e.g., aqueous
hydrochloric acid) results in the hydrolysis of the silyl ester
providing acid 8. Selective reduction of the carboxylic acid of 8
using, for example, borane-dimethylsuflide complex in a solvent
(e.g., THF) provides an intermediate alcohol. This intermediate
alcohol could be oxidized to aldehyde 9 by a number of known
methods, including, but not limited to, Swern oxidation,
Dess-Martin periodinane oxidation, Moffatt oxidation and the like.
##STR12##
The aldehyde starting materials 2 or 9 can be reductively aminated
to provide secondary or tertiary amines. This is illustrated by the
reaction of methyl 4-formyl cinnamate 2 with tryptamine 10 using
sodium triacetoxyborohydride (NaBH(OAc).sub.3) as the reducing
agent in dichloroethane (DCE) as solvent to provide amine 11. Other
reducing agents can be used, e.g., sodium borohydride (NaBH.sub.4)
and sodium cyanoborohydride (NaBH.sub.3 CN), in other solvents or
solvent mixtures in the presence or absence of acid catatylysts
(e.g., acetic acid and trifluoroacetic acid). Amine 11 can be
converted directly to hydroxamic acid 12 by treatment with 50%
aqueous hydroxylamine in a suitable solvent (e.g., THF in the
presence of a base, e.g., NaOH). Other methods of hydroxamate
formation are known and include reaction of an ester with
hydroxylamine hydrochloride and a base (e.g., sodium hydroxide or
sodium methoxide) in a suitable solvent or solvent mixture (e.g.,
methanol, ethanol or methanol/THF). ##STR13##
Aldehyde 2 can be reductively aminated with a variety of amines,
exemplified by, but not limited to, those illustrated in Table 1.
The resulting esters can be converted to target hydroxamates by the
methods listed.
TABLE 1 ##STR14## Reducing Hydroxamate Amine Conditions Conditions
R ##STR15## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR16## ##STR17## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR18## ##STR19## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR20## ##STR21## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR22## ##STR23## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR24## ##STR25## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR26## ##STR27## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR28## ##STR29## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR30## ##STR31## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
##STR32## Ph(CH.sub.2).sub.3 NH.sub.2 NaBH.sub.3 CN/MeOH/
Ph(CH.sub.2).sub.3 HOAc
An alternate synthesis of the compounds of this invention starts by
reductive amination of 4-formyl cinnamic acid 3, illustrated below
with 3-phenylpropylamine 13, using, for example, NaBH.sub.3 CN as
the reducing agent in MeOH and HOAc as a catalyst. The basic
nitrogen of the resulting amino acid 14 can be protected, for
example, as t-butoxycarbamate (BOC) by reaction with
di-t-butyldicarbonate to give 15. ##STR33##
The carboxylic acid can be coupled with a protected hydroxylamine
(e.g., O-trityl hydroxylamine) using a dehydrating agent (e.g.,
1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDCI))
and a catalyst (e.g., 1-hydroxybenzotriazole hydrate (HOBT)) in a
suitable solvent (e.g., DMF) to produce 16. Treatment of 16 with a
strong acid (e.g., trifluoroacetic acid (TFA)) provides a
hydroxamic acid 17 of the present invention. Additional examples of
compounds that can be prepared by this method are: ##STR34##
Tertiary amine compounds can be prepared by a number of methods.
Reductive amination of 30 with nicotinaldehyde 32 using NaBH.sub.3
CN as the reducing agent in dichloroethane and HOAc as a catalyst
provides ester 34. Other reducing agents can be used (e.g.,
NaBH.sub.4 and NaBH(OAc).sub.3) in other solvents or solvent
mixtures in the presence or absence of acid catalysts (e.g., acetic
acid, trifluoroacetic acid and the like). Reaction of ester 34 with
HONH.sub.2.HCl, NaOH in MeOH provides hydroxamate 36. ##STR35##
Tertiary amine compounds prepared by this methodology are
exemplified, but not limited to, those listed in Table 2.
TABLE 2 ##STR36## ##STR37## Hydroxamate Reducing Conditions
Conditions ##STR38## NaBH(OAc).sub.3 HOAc, DCE HONH.sub.2.HCl/
NaOMe/MeOH ##STR39## NaBH(OAc).sub.3 HOAc, DCE HONH.sub.2.HCl/
NaOMe/MeOH ##STR40## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in
MeOH ##STR41## NaBH.sub.3 CN/MeOH/ HOAc 2M HONH.sub.2 in MeOH
##STR42## NaBH(OAc).sub.3 HOAc, DCE 2M HONH.sub.2 in MeOH
An alternate method for preparing tertiary amines is by reacting a
secondary amine with an alkylating agent in a suitable solvent in
the presence of a base. For example, heating a dimethylsulfoxide
(DMSO) solution of amine 11 and bromide 40 in the presence of
(i-Pr).sub.2 NEt yielded tertiary amine 42. Reaction of the
tertiary amine 42 with HONH.sub.2.HCl, NaOH in MeOH provides
hydroxamate 43. The silyl group can be removed by any method known
to those skilled in the art. For example, the hydroxamate 43 can be
treated with an acid, e.g., trifluoroacetic acid, or fluoride to
produce hydroxyethyl compound 44. ##STR43##
The hydroxamate compound, or salt thereof, is suitable for
preparing pharmaceutical compositions, especially pharmaceutical
compositions having deacetylase, especially histone deacetylase,
inhibiting properties. Studies with athymic mice demonstrate that
the hydroxamate compound causes HDA inhibition and increased
histone acetylation in vivo, which triggers changes in gene
expression that correlate with tumor growth inhibition.
The present invention further includes pharmaceutical compositions
comprising a pharmaceutically effective amount of one or more of
the above-described compounds as active ingredient. Pharmaceutical
compositions according to the invention are suitable for enteral,
such as oral or rectal, and parenteral administration to mammals,
including man, for the treatment of tumors, alone or in combination
with one or more pharmaceutically acceptable carriers.
The hydroxamate compound is useful in the manufacture of
pharmaceutical compositions having an effective amount the compound
in conjunction or admixture with excipients or carriers suitable
for either enteral or parenteral application. Preferred are tablets
and gelatin capsules comprising the active ingredient together with
(a) diluents; (b) lubricants, (c) binders (tablets); if desired,
(d) disintegrants; and/or (e) absorbents, colorants, flavors and
sweeteners. Injectable compositions are preferably aqueous isotonic
solutions or suspensions, and suppositories are advantageously
prepared from fatty emulsions or suspensions. The compositions may
be sterilized and/or contain adjuvants, such as preserving,
stabilizing, wetting or emulsifying agents, solution promoters,
salts for regulating the osmotic pressure and/or buffers. In
addition, the compositions may also contain other therapeutically
valuable substances. The compositions are prepared according to
conventional mixing, granulating or coating methods, respectively,
and contain preferably about 1 to 50% of the active ingredient.
Suitable formulations also include formulations for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain antioxidants, buffers, bacteriostats
and solutes which render the formulation isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit-dose or
multi-dose containers, for example, sealed ampules and vials, and
may be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid carrier, for example, water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
As discussed above, the compounds of the present invention are
useful for treating proliferative diseases. A proliferative disease
is mainly a tumor disease (or cancer) (and/or any metastases). The
inventive compounds are particularly useful for treating a tumor
which is a breast cancer, genitourinary cancer, lung cancer,
gastrointestinal cancer, epidermoid cancer, melanoma, ovarian
cancer, pancreas cancer, neuroblastoma, head and/or neck cancer or
bladder cancer, or in a broader sense renal, brain or gastric
cancer; in particular (i) a breast tumor; an epidermoid tumor, such
as an epidermoid head and/or neck tumor or a mouth tumor; a lung
tumor, for example a small cell or non-small cell lung tumor; a
gastrointestinal tumor, for example, a colorectal tumor; or a
genitourinary tumor, for example, a prostate tumor (especially a
hormone-refractory prostate tumor); or (ii) a proliferative disease
that is refractory to the treatment with other chemotherapeutics;
or (iii) a tumor that is refractory to treatment with other
chemotherapeutics due to multidrug resistance.
In a broader sense of the invention, a proliferative disease may
furthermore be a hyperproliferative condition such as leukemias,
hyperplasias, fibrosis (especially pulmonary, but also other types
of fibrosis, such as renal fibrosis), angiogenesis, psoriasis,
atherosclerosis and smooth muscle proliferation in the blood
vessels, such as stenosis or restenosis following angioplasty.
Where a tumor, a tumor disease, a carcinoma or a cancer are
mentioned, also metastasis in the original ,organ or tissue and/or
in any other location are implied alternatively or in addition,
whatever the location of the tumor and/or metastasis.
The compound is selectively toxic or more toxic to rapidly
propiferating cells than to normal cells, particularly in human
cancer cells, e.g., cancerous tumors, the compound has significant
antiproliferative effects and promotes differentiation, e.g., cell
cycle arrest and apoptosis. In addition, the hydroxamate compound
induces p21, cyclin-CDK interacting protein, which induces either
apoptosis or G1 arrest in a variety of cell lines.
The following examples are intended to illustrate the invention and
are not to be construed as being limitations thereto.
EXAMPLE P1
Preparation of
N-Hydroxy-3-[4-[[[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-prope
namide
4-formylcinnamic acid methylester is produced by adding
4-formylcinnamic acid (25 g, 0.143 mol) in MeOH and HCl (6.7 g,
0.18 mol). The resulting suspension is heated to reflux for 3
hours, cooled and evaporated to dryness. The resulting yellow solid
is dissolved in EtOAc, the solution washed with saturated
NaHCO.sub.3, dried (MgSO.sub.4) and evaporated to give a pale
yellow solid which is used without further purification (25.0 g,
92%). To a solution of tryptamine (16.3 g, 100 mmol) and
4-formylcinnamic acid methylester (19 g, 100 mmol) in
dichloroethane, NaBH(OAc).sub.3 (21 g, 100 mmol) is added. After 4
hours the mixture is diluted with 10% K.sub.2 CO.sub.3 solution,
the organic phase separated and the aqueous solution extracted with
CH.sub.2 Cl.sub.2. The combined organic extracts are dried
(Na.sub.2 SO.sub.4), evaporated and the residue purified by flash
chromatography to produce
3-(4-{[2-(1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-(2E)-2-propenoic
acid methyl ester (29 g). A solution of KOH (12.9 g 87%, 0.2 mol)
in MeOH (100 mL) is added to a solution of HONH.sub.2.HCl (13.9 g,
0.2 mol) in MeOH (200 mL) and a precipitate results. After 15
minutes the mixture is filtered, the filter cake washed with MeOH
and the filtrate evaporated under vacuum to approximately 75 mL.
The mixture is filtered and the volume adjusted to 100 mL with
MeOH. The resulting solution 2M HONH.sub.2 is stored under N.sub.2
at -20.degree. C. for up to 2 weeks. Then
3-(4-{[2-(1H-indol-3-yl-ethylamino]-methyl}-phenyl)-(2E)-2-propenoic
acid methyl ester (2.20 g, 6.50 mmol) is added to 2 M HONH.sub.2 in
MeOH (30 mL, 60 mmol) followed by a solution of KOH (420 mg, 6.5
mmol) in MeOH (5 mL). After 2 hours dry ice is added to the
reaction and the mixture is evaporated to dryness. The residue is
dissolved in hot MeOH (20 mL), cooled and stored at -20.degree. C.
overnight. The resulting suspension is filtered, the solids washed
with ice cold MeOH and dried under vacuum, producing
N-Hydroxy-3-[4-[[[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-prope
namide (m/z 336 [MH.sup.+ ]).
EXAMPLE P2
Preparation of
N-Hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)-ethyl]-amino]methyl]ph
enyl]-2E-2-propenamide
A solution of
3-(4-{[2-(1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-(2E)-2-propenoic
acid methyl ester (12.6 g, 37.7 mmol),
(2-bromoethoxy)-tert-butyldimethylsilane (12.8 g, 53.6 mmol),
(i-Pr).sub.2 NEt, (7.42 g, 57.4 mmol) in DMSO (100 mL) is heated to
50.degree. C. After 8 hours the mixture is partitioned with
CH.sub.2 Cl.sub.2 /H.sub.2 O. The organic layer is dried (Na.sub.2
SO.sub.4) and evaporated. The residue is chromatographed on silica
gel to produce
3-[4-({[2-(tert-butyldimethylsilanyloxy)-ethyl]-[2-(1H-indol-3-yl)-ethyl]-
amino}-methyl)-phenyl]-(2E)-2-propenoic acid methyl ester (13.1 g).
Following the procedure described for the preparation of the
hydroxamate compound in Example P1,
3-[4-({[2-(tert-butyldimethylsilanyloxy)-ethyl]-[2-(1H-indol-3-yl)-ethyl]-
amino}-methyl)-phenyl]-(2E)-2-propenoic acid methyl ester (5.4 g,
11 mmol) is converted to
N-hydroxy-3-[4-({[2-(tert-butyldimethylsilanyloxy)-ethyl]-[2-(1H-indol-3-y
l)-ethyl]-amino}-methyl)-phenyl]-(2E)-2-propenamide (5.1 g,) and
used without further purification. The hydroxamic acid (5.0 g, 13.3
mmol) is then dissolved in 95% TFA/H.sub.2 O (59 mL) and heated to
40-50.degree. C. for 4 hours. The mixture is evaporated and the
residue purified by reverse phase HPLC to produce
N-Hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)-ethyl]-amino]methyl]ph
enyl]-2E-2-propenamide as the trifluoroacetate salt (m/z 380
[MH.sup.+ ]).
EXAMPLE P3
Preparation of
N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2
E-2-propenamide
A suspension of LiAlH.sub.4 (17 g, 445 mmol) in dry THF(1000 mL) is
cooled to 0.degree. C. and 2-methylindole-3-glyoxylamide (30 g, 148
mmol) is added in portions over 30 min. The mixture is stirred at
room temperature for 30 min. and then maintained at reflux for 3 h.
The reaction is cooled to 0.degree. C. and treated with H.sub.2 O
(17 ml), 15% NaOH (aq., 17 ml) and H.sub.2 O (51 ml). The mixture
is treated with MgSO.sub.4, filtered and the filtrate evaporated to
give 2-methyltryptamine which is dissolved in MeOH. Methyl
4-formylcinnamate (16.9 g, 88.8 mmol) is added to the solution,
followed by NaBH.sub.3 CN (8.4 g) and AcOH (1 equiv.). After 1 h
the reaction is diluted with NaHCO.sub.3 (aq.) and extracted with
EtOAc. The organic extracts are dried (MgSO.sub.4), filtered and
evaporated. The residue is purified by chromatography to give
3-(4-{[2-(2-methyl-1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-(2E)-2-prope
noic acid methyl ester. The ester is dissolved in MeOH, 1.0 M
HCl/dioxane (1-1.5 eqiv.) is added followed by Et.sub.2 O. The
resulting precipitate is filtered and the solid washed with
Et.sub.2 O and dried thoroughly to give
3-(4-{[2-(2-methyl-1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-(2
E)-2-propenoic acid methyl ester hydrochloride. 1.0 M NaOH (aq., 85
mL) is added to an ice cold solution of the methyl ester
hydrochloride (14.9 g, 38.6 mmol) and HONH.sub.2 (50% aq. solution,
24.0 mL, ca. 391.2 mmol). After 6 h, the ice cold solution is
diluted with H.sub.2 O and NH.sub.4 Cl (aq., 0.86 M, 100 mL). The
resulting precipitate is filtered, washed with H.sub.2 O and dried
to afford
N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2
E-2-propenamide (m/z 350 [MH.sup.+ ]).
EXAMPLES 1-265
The following compounds are prepared by methods analogous to those
disclosed in Examples P1, P2 and P3:
m/z Example STRUCTURE (MH.sup.+) 1 ##STR44## 426 2 ##STR45## 3
##STR46## 4 ##STR47## 325 5 ##STR48## 6 ##STR49## 7 ##STR50## 8
##STR51## 465 9 ##STR52## 10 ##STR53## 11 ##STR54## 12 ##STR55##
420 13 ##STR56## 420 14 ##STR57## 15 ##STR58## 465 16 ##STR59## 385
17 ##STR60## 550 18 ##STR61## 432 19 ##STR62## 366 20 ##STR63## 350
21 ##STR64## 22 ##STR65## 442 23 ##STR66## 338 24 ##STR67## 464 25
##STR68## 541 26 ##STR69## 27 ##STR70## 28 ##STR71## 417 29
##STR72## 30 ##STR73## 31 ##STR74## 380 32 ##STR75## 436 33
##STR76## 34 ##STR77## 493 35 ##STR78## 477 36 ##STR79## 586 37
##STR80## 513 38 ##STR81## 378 39 ##STR82## 408 40 ##STR83## 449 41
##STR84## 438 42 ##STR85## 452 43 ##STR86## 507 44 ##STR87## 565 45
##STR88## 46 ##STR89## 47 ##STR90## 48 ##STR91## 49 ##STR92## 50
##STR93## 51 ##STR94## 470 52 ##STR95## 53 ##STR96## 548 54
##STR97## 623 55 ##STR98## 456 56 ##STR99## 478 57 ##STR100## 394
58 ##STR101## 422 59 ##STR102## 479 60 ##STR103## 603 61 ##STR104##
477 62 ##STR105## 539 63 ##STR106## 523 64 ##STR107## 65 ##STR108##
66 ##STR109## 67 ##STR110## 68 ##STR111## 539 69 ##STR112## 495 70
##STR113## 71 ##STR114## 379 72 ##STR115## 478 73 ##STR116## 462 74
##STR117## 378 75 ##STR118## 76 ##STR119## 493 77 ##STR120## 503 78
##STR121## 350 79 ##STR122## 549 80 ##STR123## 471 81 ##STR124##
350 82 ##STR125## 418 83 ##STR126## 486 84 ##STR127## 524 85
##STR128## 424 86 ##STR129## 364 87 ##STR130## 440 88 ##STR131##
420 89 ##STR132## 390 90 ##STR133## 91 ##STR134## 92 ##STR135## 484
93 ##STR136## 498 94 ##STR137## 490 95 ##STR138## 96 ##STR139## 475
97 ##STR140## 525 98 ##STR141## 422 99 ##STR142## 528 100
##STR143## 448 101 ##STR144## 437 102 ##STR145## 451 103 ##STR146##
505 104 ##STR147## 519 105 ##STR148## 514 106 ##STR149## 507 107
##STR150## 626 108 ##STR151## 499 109 ##STR152## 110 ##STR153## 111
##STR154## 429 112 ##STR155## 464 113 ##STR156## 432 114 ##STR157##
422 115 ##STR158## 390 116 ##STR159## 501 117 ##STR160## 484 118
##STR161## 119 ##STR162## 587 120 ##STR163## 602 121 ##STR164## 539
122 ##STR165## 123 ##STR166## 528 124 ##STR167## 487 125 ##STR168##
126 ##STR169## 556 127 ##STR170## 128 ##STR171## 129 ##STR172## 552
130 ##STR173## 519 131 ##STR174## 450 132 ##STR175## 464 133
##STR176## 558 134 ##STR177## 533 135 ##STR178## 136 ##STR179## 527
137 ##STR180## 381 138 ##STR181## 364 139 ##STR182## 140 ##STR183##
448 141 ##STR184## 558 142 ##STR185## 143 ##STR186## 427 144
##STR187##
145 ##STR188## 432 146 ##STR189## 384 147 ##STR190## 354 148
##STR191## 149 ##STR192## 150 ##STR193## 151 ##STR194## 152
##STR195## 153 ##STR196## 154 ##STR197## 350 155 ##STR198## 366 156
##STR199## 408 157 ##STR200## 322 158 ##STR201## 364 159 ##STR202##
364 160 ##STR203## 378 161 ##STR204## 350 162 ##STR205## 463 163
##STR206## 164 ##STR207## 381 165 ##STR208## 463 166 ##STR209## 476
167 ##STR210## 168 ##STR211## 169 ##STR212## 170 ##STR213## 368 171
##STR214## 493 172 ##STR215## 527 173 ##STR216## 515 174 ##STR217##
323 175 ##STR218## 540 176 ##STR219## 441 177 ##STR220## 276 178
##STR221## 179 ##STR222## 455 180 ##STR223## 181 ##STR224## 336 182
##STR225## 347 183 ##STR226## 447 184 ##STR227## 185 ##STR228## 420
186 ##STR229## 424 187 ##STR230## 422 188 ##STR231## 189 ##STR232##
398 190 ##STR233## 418 191 ##STR234## 350 192 ##STR235## 193
##STR236## 352 194 ##STR237## 499 195 ##STR238## 408 196 ##STR239##
394 197 ##STR240## 499 198 ##STR241## 199 ##STR242## 200 ##STR243##
350 201 ##STR244## 202 ##STR245## 203 ##STR246## 204 ##STR247## 365
205 ##STR248## 465 206 ##STR249## 207 ##STR250## 410 208 ##STR251##
410 209 ##STR252## 210 ##STR253## 366 211 ##STR254## 352 212
##STR255## 213 ##STR256## 368 214 ##STR257## 338 215 ##STR258## 356
216 ##STR259## 408 217 ##STR260## 368 218 ##STR261## 396 219
##STR262## 220 ##STR263## 342 221 ##STR264## 392 222 ##STR265## 412
223 ##STR266## 337 224 ##STR267## 337 225 ##STR268## 456 226
##STR269## 364 227 ##STR270## 481 228 ##STR271## 355 229 ##STR272##
312 230 ##STR273## 424 231 ##STR274## 232 ##STR275## 351 233
##STR276## 392 234 ##STR277## 235 ##STR278## 236 ##STR279## 322 237
##STR280## 238 ##STR281## 366 239 ##STR282## 240 ##STR283## 368 241
##STR284## 242 ##STR285## 406 243 ##STR286## 398 244 ##STR287## 442
245 ##STR288## 350 246 ##STR289## 364 247 ##STR290## 402 248
##STR291## 418 249 ##STR292## 364 250 ##STR293## 251 ##STR294## 408
252 ##STR295## 253 ##STR296## 254 ##STR297## 413 255 ##STR298## 405
256 ##STR299## 257 ##STR300## 394 258 ##STR301## 390 259 ##STR302##
434 260 ##STR303## 386 261 ##STR304## 368 262 ##STR305## 412 263
##STR306## 406 264 ##STR307## 265 ##STR308## 378
The compounds of Examples 1-265 show an HDA enzyme IC.sub.50 in the
range from about 0.005 to about 0.5 .mu.M.
EXAMPLE B1
Cell lines H1299 (human lung carcinoma cell) and HCT116 (colon
tumor cell) are obtained from the American Type Culture Collection,
Rockville, Md. The cell lines are free of Mycoplasma contamination
(Rapid Detection System by Gen-Probe, Inc., San Diego, Calif.) and
viral contamination (MAP testing by MA BioServices, Inc.,
Rockville, Md.). The cell lines are propagated and expanded in RPMI
1640 medium containing 10% heat-inactivated FBS (Life Technologies,
Grand Island, N.Y.). Cell expansions for implantation are performed
in cell factories (NUNC, purchased from Fisher Scientific,
Springfield, N.J.). Cells are harvested at 50-90% confluency,
washed once with HBSS (Hank's Balanced Salt Solution) containing
10% FBS, and suspended in 100% HBSS.
Cell proliferation is measured with a commercial MTS kit (Promega,
Madision, Wis.) assay using an adaptation of published procedures,
for example, that disclosed in Feasibility of drug screening with
panels of human tumor cell lines using a microculture tetrazolium
assay, Alley M C, et al., Cancer Res. 1988; 48:589-601. Cells are
plated in 96-well tissue culture dishes, with top and bottom rows
left empty. H1299 and HCT116 cells are suspended in complete media
at a density of 5.3.times.10.sup.3 and 3.6.times.10.sup.3 cell/mL,
respectively, and 190 .mu.l are added per well. Each cell line is
added to one half of the plate. Complete medium (200 .mu.L) is
added to the top and bottom rows. Twenty-four hours later, 10 .mu.l
of MTS solution is added to one of the plates to determine the
activity at the time of compound addition (T.sub.0). The plate is
incubated at 37.degree. C. for 4 hours and the OD.sub.490 is
measured on a Molecular Devices Thermomax at 490 nm using the
Softmax program. The T.sub.0 plate serves as a reference for
initial activity at the beginning of the experiment.
Five serial dilutions (1:4) of each compound are made in a 96-deep
well plate with the highest concentrations on the edge of plate.
Two cell lines are tested with two compounds per plate.0 Ten
microliters of each of the five dilutions are added in triplicate
and complete medium alone is added to columns six and seven. The
plates are incubated at 37.degree. C. for 72 hours. The MTS
solution is added (as for the T.sub.0 plate) and read four hours
later.
In order to analyze the data, the average background value (media
alone) is subtracted from each experimental well; the triplicate
values are averaged for each compound dilution. The following
formulas are used to calculate percent growth.
in which T.sub.0 =(average value of cell viability at time
0)-background GC=average value of untreated cells (in
triplicate)-background X=average value of compound treated cells
(in triplicate)-background
The "% Growth" is plotted against compound concentration and used
to calculate IC.sub.50 s employing the linear regression techniques
between data points to predict the concentration of compounds at
50% inhibition.
Lactate salts of
N-hydroxy-3-[4-[[[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-prope
namide (CMD1),
N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phe
nyl]-2E-2-propenamide (CMD2),
N-hydroxy-3-[4-[[[2-(5-methoxy-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-
2E-2-propenamide (CMD3),
N-hydroxy-3-[4-[[[2-(5-fluoro-1H-indol-3yl)-ethyl]-amino]methyl]phenyl]-2E
-2-propenamide (CMD4),
N-hydroxy-3-[4-[[[2-(benzofur-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-prope
namide (CMD5) having a purity of higher than 95% are dissolved in
pure dimethylsulfoxide (DMSO) to create a stock solution. The stock
solution is diluted with 5% dextrose injection, USP, just prior to
dosing. In addition,
N-(2-aminophenyl)-4[N-pyridin-3-yl)methoxycarbonylaminomethyl]benzamide
is synthesized in accordance with Example 48 of EP 0 847 992 and
used as a control compound (CMDC). Inhibition of cell growth in
monolayer for 72 hours of compound treatment is measured in
triplicate experiments and used to derive the IC.sub.50 by MTS
assay. The results are shown in Table B1.
TABLE B1 Active Monolayer Growth IC.sub.50 (.mu.M) Compound H1299
HCT116 CMD1 0.40 0.03 CMD2 0.15 0.01 CMD3 0.58 0.03 CMD4 0.28 0.03
CMD5 0.18 0.03 CMDC 6.8 0.67
The results show that the hydroxamate compounds of the present
invention are highly active in inhibition of tumor cell growth. In
addition to the above results, it has been observed that the
compounds selectively inhibited tumor cells while showing minimal
inhibition activities in non-tumorous cells.
The cells treated with the hydroxamate compounds are also tested
for the induction of p21 promoter, which is a key mediator of G1
arrest and differentiation. The hydroxamate compounds activate the
p21 promoter to a readily detectable level at a concentration
within two-fold of their respective IC.sub.50 for monolayer cell
growth inhibition in H1299. Without being bound by amy particular
theory, the correlation appears to demonstrate that HDA inhibition
leads to transcriptional activation of genes that inhibit tumor
cell proliferation.
EXAMPLE B2
HDA is partially purified from H1299, human non-small cell lung
carcinoma cells (obtained from American Type Culture Collection,
12301 Parklawn Drive, Rockville, Md. 20852, USA). Cells are grown
to 70-80% confluence in RPMI media in the presence of 10% FCS,
harvested and lysed by sonication. The lysate is centrifuged at
23,420 g for 10-15 min, the supernatant is applied to a Hiload
26/10 High performance Q-sepharose column (Amersham Pharmacia
Biotech), and equilibrated with a buffer containing 20 mM Tris
pH8.0, 1 mM EDTA, 10 mM NH.sub.4 Cl.sub.2, 1 mM
.mu.-Mercaptoethanol, 5% glycerol, 2 .mu.g/mL aprotinin, 1 .mu.g/mL
leupeptin, and 400 mM PMSF. Proteins are eluted in 4 mL aliquots
with a linear gradient from 0-500 mM NaCl in the above buffer at a
flow rate of 2.5 mL/min. Each preparation of partially purified HDA
enzyme is titrated to determine the optimal amount needed to obtain
a signal to noise ratio of at least 5 to 1. Generally, 20-30 .mu.l
of partially purified HDA (5-10 mg protein/mL) is mixed with 2
.mu.L of compound solution in DMSO in a deep well titer plate
(Beckman). The compounds are serially diluted in DMSO to generate
stocks at 20-fold of the assay concentrations. Final concentrations
of compounds in the assay are 10 .mu.M, 2 .mu.M, 400 nM, 80 nM, and
16 nM with the final percentage of DMSO in each enzyme reaction
equaling 0.1%. Each concentration of compound is assayed in
duplicate. The substrate used in the reaction is a peptide of amino
acid sequence, SGRGKGGKGLGKGGAKRHRKVLRD, corresponding to the
twenty-four N-terminal amino acids of human histone H4,
biotinylated at the N-terminus and penta-acetylated, at each lysine
residue with .sup.3 H-acetate. To initiate the reaction, the
substrate is diluted in 10 .mu.L of Buffer A (100 mM Tris pH 8.0, 2
mM EDTA), added to the enzyme mixture and collected at the bottom
of the deep well plate by centrifugation for 5 minutes at 1500 rpm.
Following centrifugation, the mixture is incubated at 37.degree. C.
for 1.5 hr. The reaction is stopped by the addition of 20 .mu.L of
the Stop Buffer (0.5N HCl, 0.08M Acetic Acid). At this point, the
assay proceeds to the robotic extraction phase or is frozen for
several days at -80.degree. C.
The extraction of enzymatically cleaved .sup.3 H-acetate groups
from the reaction mixture is achieved with the solvent TBME
(t-butyl methyl ether) using the Tomtec Quadra 96 workstation. A
program is written to add 200 .mu.L of TBME to a 96 "deep well"
plate. The workstation is programmed to aspirate 50 .mu.L of air
followed by 200 .mu.L of TBME and finally another 25 .mu.L of air,
which is dispensed into the each well of the plate. The contents of
the deep well were mixed thoroughly by pipetting 160 .mu.L up and
down 10 times. Before addition of TBME to the reaction mixture, it
is necessary to "pre-wet" the pipette tips with TBME to prevent the
solvent from dripping during the transfer to the deep well plate.
The organic and aqueous phases in the deep well are separated by
centrifugation at 1500 rpm for 5 min. Opti-Phase Supermix liquid
scintillation cocktail (200 .mu.L) (Wallac) is added to each well
of the 96-well Trilux plate (Wallac). The deep well and Trilux
plates are placed back on the workstation programmed to aspirate 25
.mu.L of air into the pipette tips followed by 100 .mu.L of the
upper TBME phase and transfer it into the Trilux plate. The
solutions are mixed by pipetting and expelling 50 .mu.L, five
times, within the same well. The Trilux plate is covered with clear
film and read on a 1450 MicroBeta Trilux liquid scintillation and
luminescence counter (Wallac) with a color/chemical quench and dpm
correction.
In order to determine the IC.sub.50 values, the data are analyzed
on a spreadsheet. The analysis requires a correction for the
background luminescence that is accomplished by subtracting the dpm
values of wells without .sup.3 H substrate from the experimental
wells. The corrected dpm values along with the concentrations of
the compounds are used to calculate IC.sub.50 using the
user-defined spline function. This function utilizes linear
regression techniques between data points to calculate the
concentration of compounds that produced 50% inhibition. The
results are shown in Table B2.
TABLE B2 Compound HDA Enzyme Activity IC.sub.50 (.mu.M) CMD1 0.032
CMD2 0.063 CMD3 0.014 CMD4 0.014 CMD5 0.016 CMDC >10
EXAMPLE B3
The A549 non-small cell lung human tumor cell line is purchased
from the American Type Culture Collection, Rockville, Md. The cell
line is free of Mycoplasma contamination (Rapid Detection System by
Gen-Probe, Inc., San Diego, Calif.) and viral contamination (MAP
testing by MA BioServices, Inc., Rockville, Md.). The cell line is
propagated and expanded in RPMI 1640 medium containing 10%
heat-inactivated FBS (Life Technologies, Grand Island, N.Y.). Cell
expansions for implantation are performed in cell factories (NUNC,
purchased from Fisher Scientific, Springfield, N.J.). Cells are
harvested at 50-90% confluency, washed once with HBSS containing
10% FBS, and suspended in 100% HBSS.
Outbred athymic (nu/nu) female mice ("Hsd:Athymic Nude-nu" from
Harlan Sprague Dawley, Indianapolis, Ind.) are anesthetized with
Metofane (Mallinckrodt Veterinary, Inc., Mundelein, Ill.), and 100
.mu.L of the cell suspension containing 1.times.10.sup.7 cells is
injected subcutaneously into the right axillary (lateral) region of
each animal. Tumors are allowed to grow for about 20 days until a
volume of .about.100 mm.sup.3 is achieved. At this point, mice
bearing tumors with acceptable morphology and size are sorted into
groups of eight for the study. The sorting process produces groups
balanced with respect to mean and range of tumor size. Antitumor
activity is expressed as % T/C, comparing differences in tumor
volumes for treatment group (T) to vehicle control group (C).
Regressions are calculated using the formula:
(1-T/T.sub.0).times.100%, where T is the tumor volume for the
treatment group at the end of the experiment, and To is the tumor
volume at the beginning of the experiment.
CMD1 is administered intravenously, once daily 5.times./week for
three weeks, at doses of 10, 25, 50, or 100 mg/kg. The final DMSO
concentration is 10%. Each test group has eight mice. Tumors are
measured, and individual animal body weights recorded. Table B3
shows the results on the 41.sup.st day.
TABLE B3 .DELTA. MEAN .DELTA. % TUMOR BODY DOSE VOLUME*.sup.1
WEIGHT*.sup.2 COMPOUND (mg/kg) (mm.sup.3 .+-. SEM*.sup.3) % T/C (%
.+-. SEM*.sup.3) 10% DMSO/ -- 376 .+-. 55 -- +11.9 .+-. 0.2
D5W*.sup.4 CMD1 10 121 .+-. 27 32 +1.3 .+-. 0.3 CMD1 25 77 .+-. 32
20 -0.9 .+-. 0.3 CMD1 50 57 .+-. 10 15 -0.4 .+-. 0.3 CMD1 100 28
.+-. 25 7 +0.4 .+-. 0.3 Note: *.sup.1 Difference in mean tumor
volume for a group of animals at the end of the experiment minus
mean tumor volume at the beginning. *.sup.2 Difference in body
weight for a group of animals at the end of the experiment minus
mean tumor volume at the beginning. *.sup.3 Standard error of the
mean *.sup.4 5% dextrose injection, USP.
EXAMPLE B4
Example B3 repeated except CMD2 is used. Table B4 shows the
results.
TABLE B4 .DELTA. MEAN .DELTA. % TUMOR BODY DOSE VOLUME WEIGHT
COMPOUND (mg/kg) (mm.sup.3 .+-. SEM) % T/C (% .+-. SEM) 10%
DMSO/D5W -- 135 .+-. 43 -- +6.7 .+-. 1.1 CMD2 25 37 .+-. 16 27 -4.2
.+-. 2.5 CMD2 50 29 .+-. 15 21 -2.9 .+-. 1.5
EXAMPLE B5
Example B3 is repeated except the HCT116 colon tumor cell line is
used in place of the A549 cell line. The HCT116 cell line is also
obtained from American Type Culture Collection, Rockville, Md., and
the cell line is free of Mycoplasma contamination and viral
contamination. The results are recorded on the 34.sup.th day and
are shown in Table B5.
TABLE B5 .DELTA. MEAN .DELTA. % TUMOR BODY DOSE VOLUME WEIGHT
COMPOUND (mg/kg) (mm.sup.3 .+-. SEM) % T/C (% .+-. SEM) 10%
DMSO/D5W -- 759 .+-. 108 -- -0.4 .+-. 0.4 CMD1 50*.sup.10 186 .+-.
40 25 -7.4 .+-. 0.8 CMD1 100 140 .+-. 38 18 -3.2 .+-. 0.4 Note:
*.sup.10 Seven mice are tested in this group.
EXAMPLE B6
Example B4 is repeated except the HCT116 colon tumor cell line is
used in place of the A549 cell line. The HCT116 is also obtained
from American Type Culture Collection, Rockville, Md., and the cell
line is free of Mycoplasma contamination and viral contamination.
The results are recorded on the 34.sup.th day and are shown in
Table B6.
TABLE B6 .DELTA. MEAN .DELTA. % TUMOR BODY DOSE VOLUME WEIGHT
COMPOUND (mg/kg) (mm.sup.3 .+-. SEM) % T/C (% .+-. SEM) 10%
DMSO/D5W -- 759 .+-. 108 -- -0.4 .+-. 0.4 CMD2 10 422 .+-. 75 56
-10.2 .+-. 0.5 CMD2 25 305 .+-. 47 40 -7.0 .+-. 0.2 CMD2 50 97 .+-.
30 13 -7.3 .+-. 0.3 CMD2 100 132 .+-. 30 17 -9.4 .+-. 0.4
EXAMPLE B7
Annexin V binding was used as a marker for the early stages of
apoptosis. A549, HCT116 and Normal Dermal Human Fibroblasts (NDHF)
cells are treated separately with four compounds (CMD1, CMD2, CMD3
and CMD4) for 24 or 48 hours, stained with annexin V and compared
to cells treated similarly with vehicle (DMSO). Cells are examined
by fluorescence microscopy. Those undergoing apoptosis exhibit
green fluorescent membrane staining. Viability is assessed by the
counterstain, propidium iodide. Cells detected by red fluorescence
are not viable. A small percentage of A549 and the majority of
HCT116 cells exhibit cell surface staining with annexin V after 24
hour exposure to each of the four compounds. After 48 hour
treatment, the majority of the A549 and HCT116 stain with annexin V
and/or propidium iodide indicating that the compounds induce
apoptotic cell death. In contrast, NDHF cells do not show
noticeable annexin V staining after 24 hour exposure and limited
annexin V staining with CMD3 after 48 hour. These data show that
NDHF cells predominantly underwent non-lethal growth arrest upon
compound treatment, consistent with the cell cycle profile.
The staining results demonstrate that the hydroxamate compounds of
the present invention cause tumor cells to die by apoptosis, while
causing normal fibroblast to predominantly undergo cell cycle
arrest, clearly demonstrating the selective efficacy of the present
compounds.
* * * * *